An introduction to the CRISPR/Cas9 system of genome engineering
What is CRISPR/Cas9?
The type II CRISPR/Cas system (clustered regularly interspaced short palindromic repeats/ CRISPR-associated) is used by bacteria as an RNA-guided defence system against invading viruses and plasmids (Barrangou et al., 2007). In Streptococcus pyogenes, the Cas9 endonuclease is guided to its target site by complementary base pairing of CRISPR RNAs (crRNAs) with the target DNA sequence, and requires a third component, the trans-activating CRISPR RNA (tracrRNA), which recruits the crRNA into the Cas9 complex (Brouns et al., 2008; Gasiunas et al., 2012; Jinek et al., 2012). This system has recently been adapted to target double strand breaks in the genomes of other organisms including humans (Cong et al., 2013; Mali et al., 2013), mice (Wang et al., 2013) and zebrafish (Hwang et al., 2013).
Application to Drosophila
We have modified a two-component system described previously (Cong et al., 2013; Dahlem et al., 2012; Jinek et al., 2012; Mali et al., 2013) in which the crRNA and tracrRNA are fused into a single synthetic guide RNA (sgRNA) to efficiently create targeted mutations in the Drosophila yellow and white genes by direct injection of Cas9 mRNA and a sgRNA into the embryo. We show that the double strand breaks induced can result in small insertions and deletions (indels) at the target sites due to inefficient repair by non-homologous end joining (NHEJ) (Bibikova et al., 2002). This process occurs with extremely high efficiency, with up to 88% of injected flies having detectable mutations. Mutants can also be transmitted through the germline to the following generation at a rate of up to 34.5% of total offspring. Our new method offers at least a 10-fold improvement in efficiency over a recently published report (Gratz et al., 2013, http://flycrispr.molbio.wisc.edu/), enabling more general application to gene knockouts.
Detection of indels by high resolution melt analysis (HRMA)
We also describe a system for simple and effective detection of the indels created in the injected generation (G0) by high resolution melt analysis (HRMA). This can be applied to any gene and makes it possible to follow the mutations generated. It is highly sensitive, and capable of detecting a single base deletion in a heterozygous fly.